Epo Dependent Signaling In Macrophages Regulates Stress Erythropoiesis

Abstract Steady State erythropoiesis occurs in the bone marrow and is primarily homeostatic. In response to anemic stress the need for new erythrocytes quickly outpaces the erythropoietic capacity of steady state erythropoiesis. At these times, stress erythropoiesis predominates. Stress erythropoiesis is best understood in mice where this process is primarily extra-medullary occurring in the adult spleen and liver and in the fetal liver during development. Stress erythropoiesis utilizes progenitors and signals that are distinct from steady state erythropoiesis. Using a variety of experimental systems, we have developed a model for stress erythropoiesis during the recovery from anemic stress. This recovery can be divided into three stages. Amplification of progenitors that exhibit stem cell properties, the induction of a signal that promotes the switch from amplifying stress progenitors to differentiating stress progenitors and the final stage where stress progenitors rapidly differentiate into new erythrocytes. We have identified specific stress progenitor populations at each stage on this process as well as the signals that regulate the amplification, the switch to differentiation and differentiation of stress erythroid progenitors. Here we show that macrophage dependent signals play key roles at each stage of stress erythropoiesis. The transition from amplifying stress erythroid progenitors to differentiating stress erythroid progenitors is mediated by Epo dependent signaling in macrophages which changes the signals made by the macrophage microenvironment from those that promote amplification (Wnt family factors) to those that promote differentiation (PGE2). This paradigm is true for murine and human stress erythroid progenitors. This analysis reveals a dynamic interplay between progenitor cells, the macrophage microenvironment and hypoxic tissues in vivo during the recovery from anemic stress. Disclosures: No relevant conflicts of interest to declare.

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BMP4/Madh5 Dependent Signaling Is Required for the Expansion of a Population of Stress Erythroid Progenitors in the Fetal Liver.

Blood ◽

10.1182/blood.v106.11.4195.4195 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4195-4195

Author(s):

Robert F. Paulson ◽

Prashanth Porayette

Keyword(s):

Bone Marrow ◽

Steady State ◽

Fetal Liver ◽

Erythroid Progenitors ◽

Acute Anemia ◽

Erythroid Response ◽

Stress Erythropoiesis ◽

And Control ◽

Flexed Tail

Abstract Fetal liver hematopoiesis is primarily erythropoiesis, which robustly produces erythrocytes to meet the growing need of the developing embryo. In many ways fetal liver erythropoiesis resembles stress erythropoiesis in the adult, where in response to acute anemia, a unique population of stress erythroid progenitors is rapidly expanded in the spleen. The development of these stress progenitors requires BMP4/Madh5 dependent signals. Spleen stress progenitors exhibit properties that are distinct from bone marrow steady state progenitors in that they are able to rapidly form large BFU-E colonies, which require only Epo stimulation for their generation. Mice mutant at the flexed-tail locus exhibit a defective stress erythroid response because of a mutation in Madh5. In addition to this defect, flexed-tail mice also exhibit a severe fetal-neonatal anemia. We have analyzed fetal liver erythropoiesis in flexed-tail and control embryos. We show that BMP4 is expressed in the fetal liver and its expression correlates with the time of maximum erythropoiesis. In flexed-tail mutant embryos the expression is delayed and this correlates with both a delay and a defect in the expansion of erythroid progenitors. Our analysis also shows that the fetal liver contains two types of erythroid progenitors. One type exhibits the properties of stress BFU-E found in the adult spleen, which are compromised in flexed-tail embryos and a second type that is similar to bone marrow steady state BFU-E. These data demonstrate that BMP4 dependent signaling drives the expansion of erythroid progenitors in the fetal liver in a manner similar to stress erythropoiesis in the adult spleen.

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Functional Requirements of a Samd14-Capping Protein Interaction in Stress Erythropoiesis

Blood ◽

10.1182/blood-2021-152898 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 288-288

Author(s):

Suhita Ray ◽

Linda Chee ◽

Nicholas T. Woods ◽

Kyle J Hewitt

Keyword(s):

Steady State ◽

Hemolytic Anemia ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Interacting Proteins ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Capping Protein ◽

Sam Domain ◽

Stress Erythropoiesis

Abstract Stress erythropoiesis describes the process of accelerating red blood cell (RBC) production in anemia. Among a number of important mediators of stress erythropoiesis, paracrine signals - involving cooperation between SCF/c-Kit signaling and other signaling inputs - are required for the activation/function of stress erythroid progenitors. Whereas many critical factors required to drive erythropoiesis in normal physiological conditions have been described, whether distinct mechanisms control developmental, steady-state, and stress erythropoiesis in anemia is poorly understood. Our prior work revealed that the Sterile Alpha Motif (SAM) Domain 14 (Samd14) gene is transcriptionally upregulated in a model of acute hemolytic anemia induced by the RBC-lysing chemical phenylhydrazine. Samd14 is regulated by GATA binding transcription factors via an intronic enhancer (Samd14-Enh). In a mouse knockout of Samd14-Enh (Samd14-Enh -/-), we established that the Samd14-Enh is dispensable for steady-state erythropoiesis but is required for recovery from severe hemolytic anemia. Samd14 promotes c-Kit signaling in vivo and ex vivo, and the SAM domain of Samd14 facilitates c-Kit-mediated cellular signaling and stress progenitor activity. In addition, the Samd14 SAM domain is functionally distinct from closely related SAM domains, which demonstrates a unique role for this SAM domain in stress signaling and cell survival. In our working model, Samd14-Enh is part of an ensemble of anemia-responsive enhancers which promote stress erythroid progenitor activity. However, the mechanism underlying Samd14's role in stress erythropoiesis is unknown. To identify potential Samd14-interacting proteins that mediate its function, we performed immunoprecipitation-mass spectrometry on the Samd14 protein. We found that Samd14 interacted with α- and β heterodimers of the F-actin capping protein (CP) complex independent of the SAM domain. CP binds to actin during filament assembly/disassembly and plays a role in cell morphology, migration, and signaling. Deleting a 17 amino acid sequence near the N-terminus of Samd14 disrupted the Samd14-CP interaction. However, mutating the canonical RxR of the CP interaction (CPI) motif, which is required for CP-binding in other proteins, does not abrogate the Samd14-CP interaction. Moreover, replacing this sequence with the canonical CPI domain of CKIP-1 completely disrupts the interaction, indicating that other sequence features are required to maintain the Samd14-CP complex. Ex vivo knockdown of the β-subunit of CP (CPβ), which disrupts the integrity of the CP complex, decreased the percentage of early erythroid precursors (p<0.0001) and decreased (3-fold) progenitor activity as measured by colony formation assays (similar to knockdown of Samd14). Taken together, these data indicate that Samd14 interacts with CP via a unique CP binding (CPB) domain, and that the CP complex coordinates erythroid differentiation in stress erythroid progenitors. To test the function of the Samd14-CP complex, we designed an ex vivo genetic complementation assay to express Samd14 lacking the CPB-domain (Samd14∆CPB) in stress erythroid progenitors isolated from anemic Samd14-Enh -/- mice. Phospho-AKT (Ser473) and phospho-ERK (Thr202/Tyr204) levels in Samd14∆CPB were, respectively, 2.2 fold (p=0.007) and ~7 fold (n=3) lower than wild type Samd14 expressing cells, 5 min post SCF stimulation. Relative to Samd14, Samd14∆CPB expression reduced burst forming unit-erythroid (BFU-E) (2.0 fold) and colony forming unit-erythroid (CFU-E) (1.5 fold). These results revealed that the Samd14-CP interaction is a determinant of BFU-E and CFU-E progenitor cell levels and function. Remarkably, as the requirement of the CPB domain in BFU-E and CFU-E progenitors is distinct from the Samd14-SAM domain (which promotes BFU-E but not CFU-E), the function of Samd14 in these two cell types may differ. Ongoing studies will examine whether the function of Samd14 extends beyond SCF/c-Kit signaling and establish cell type-dependent functions of Samd14 and Samd14-interacting proteins. Given the critical importance of c-Kit signaling in hematopoiesis, the role of Samd14 in mediating pathway activation, and our discovery implicating the capping protein complex in erythropoiesis, it is worth considering the pathological implications of this mechanism in acute/chronic anemia and leukemia. Disclosures No relevant conflicts of interest to declare.

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Complete Spatial Mapping of Steady-State and Stress Erythropoiesis

Blood ◽

10.1182/blood-2020-142973 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 7-7

Author(s):

Qingqing Wu ◽

Jizhou Zhang ◽

Courtney Johnson ◽

Anastasiya Slaughter ◽

Margot Lindsay May ◽

...

Keyword(s):

Bone Marrow ◽

Steady State ◽

Single Cells ◽

Differentially Expressed ◽

Published Data ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Clonal Architecture ◽

Stress Erythropoiesis

The anatomy of differentiation in the bone marrow (BM) is poorly understood due to lack of markers to image stepwise HSPC differentiation. We analyzed 250+ cell surface molecules in all hematopoietic progenitors and identified 56 differentially expressed markers in at least one HSPC that can be "mixed and matched" to prospectively image any HSPC of interest in the bone marrow. We used this data to develop a pipeline to map stepwise erythropoiesis in vivo. We found that all erythroid progenitors can be defined as Ly6C-CD27-ESAM-CD117+ cells and then Pre-MegE (earliest erythroid progenitor Cell Stem Cell. 2007 1(4):428-42) are CD150+CD71-. These give rise to CD71+CD150+ Pre-CFU-E that differentiate into CD71+CD150- CFU-E that then generate early erythroblasts. All BFU-E activity was restricted to Pre-MegE and Pre- CFU-E (70 and 30% of all BFU-E) whereas all CFU-E colonies were spread between Pre-MegE (44%), pre-CFU-E (10%) and CFU-E (46%). We also confirmed previously published data showing that CD71 and Ter119 can be used to image stepwise terminal erythropoiesis; CD71+Ter119dim early erythroblasts, CD71+Ter119bright late erythroblasts, CD71dimTer119bright reticulocytes and CD71-Ter119bright erythrocytes. Importantly, all populations were detected at identical frequencies using FACS or confocal imaging indicating that our imaging strategy detects all erythroid cells (Pre-CFU-E: 0.022 vs 0.027 %; CFUE: 0.32 vs 0.30%; Early-Ery: 0.62 vs 0.66%; Late-Ery: 32.05 vs 32.12%; Reticulocyte: 5.98 vs. 3.36%; Erythrocytes: 12.49 vs. 13.47%). We mapped the 3D location of every erythroid lineage cell in mouse sternum and interrogated the spatial relationships between the different maturation steps and with candidate niches. We compared the interactions found in vivo with those found in random simulations. Specifically, we used CD45 and Ter119 to obtain the spatial coordinates of every hematopoietic cell. Then we randomly placed each type of erythroid lineage cell at identical frequencies as those found in vivo to generate random simulations. We found erythroid progenitors show no specific association with HSC, indicating that Pre-Meg-E or more primitive progenitors leave the HSC niche after differentiation. Both Pre-Meg-E and Pre-CFU-E are found as single cells through the central BM space and do not specifically associate with other progenitors, or components of the microenvironment. In contrast almost all CFU-E locate to strings (28 strings per sternum) containing 8 CFU-E that are selectively recruited to sinusoids (mean CFU-E to sinusoid distance=2.2µm). As soon as CFU-E detach from sinusoids they downregulate CD117 and upregulate CD71 giving rise to a cluster of early erythroblasts that buds from the vessel. These progressively upregulate Ter119 to generate large clusters of late erythroblasts that in turn differentiate into clusters of reticulocytes and erythrocytes. To examine the clonal architecture of erythropoiesis we used Ubc-creERT2:confetti mice where a tamoxifen pulse leads to irreversible expression of GFP, CFP, YFP or RFP. Four weeks later we found that the CFU-E strings are oligoclonal with each clone contributing 2-6 CFU-E to the string. The budding erythroblasts clusters are similarly organized. These indicate that different CFU-E are serially recruited to the same sinusoidal spot where they self-renew 1-2 times and then undergo terminal differentiation. We then tracked how this architecture changed in response to stress (hemorrhage). Two days after bleeding we found that Pre-Meg-E and Pre-CFU-E numbers and locations were unaltered. The number of CFU-E strings remained constant (30 CFUE strings/sternum) but all strings contained more CFU-E (2-fold) suggesting increased self-renewal. Unexpectedly, fate mapping showed that the size of CFU-E clones did not increase when compared to steady-state. These results indicate that all CFU-E expand in respond to stress and that this is mediated via increased recruitment and differentiation of upstream progenitors. In summary we have found 56 differentially expressed markers that can be combined to detect most HSPC; validated a 5-color stain to image and fate map all steps of red blood cell maturation in situ; demonstrated that terminal erythropoiesis emerges from strings of sinusoidal CFU-E, and revealed the clonal architecture of normal and stress erythropoiesis. Disclosures No relevant conflicts of interest to declare.

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The MAPK ERK1 is a negative regulator of the adult steady-state splenic erythropoiesis

Blood ◽

10.1182/blood-2009-09-242487 ◽

2010 ◽

Vol 115 (18) ◽

pp. 3686-3694 ◽

Cited By ~ 31

Author(s):

Soizic Guihard ◽

Denis Clay ◽

Laurence Cocault ◽

Nathalie Saulnier ◽

Paule Opolon ◽

...

Keyword(s):

Bone Marrow ◽

Steady State ◽

Bone Marrow Cells ◽

Serum Levels ◽

Negative Regulator ◽

Erythroid Progenitors ◽

Stress Erythropoiesis ◽

Extracellular Signal ◽

Mitogen Activated Protein

Abstract The mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1 (ERK1) and ERK2 are among the main signal transduction molecules, but little is known about their isoform-specific functions in vivo. We have examined the role of ERK1 in adult hematopoiesis with ERK1−/− mice. Loss of ERK1 resulted in an enhanced splenic erythropoiesis, characterized by an accumulation of erythroid progenitors in the spleen, without any effect on the other lineages or on bone marrow erythropoiesis. This result suggests that the ablation of ERK1 induces a splenic stress erythropoiesis phenotype. However, the mice display no anemia. Deletion of ERK1 did not affect erythropoietin (EPO) serum levels or EPO/EPO receptor signaling and was not compensated by ERK2. Splenic stress erythropoiesis response has been shown to require bone morphogenetic protein 4 (BMP4)–dependent signaling in vivo and to rely on the expansion of a resident specialized population of erythroid progenitors, termed stress erythroid burst-forming units (BFU-Es). A great expansion of stress BFU-Es and increased levels of BMP4 mRNA were found in ERK1−/− spleens. The ERK1−/− phenotype can be transferred by bone marrow cells. These findings show that ERK1 controls a BMP4-dependent step, regulating the steady state of splenic erythropoiesis.

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A critical role of VLA-4 in erythropoiesis in vivo

Blood ◽

10.1182/blood.v87.6.2513.bloodjournal8762513 ◽

1996 ◽

Vol 87 (6) ◽

pp. 2513-2517 ◽

Cited By ~ 54

Author(s):

K Hamamura ◽

H Matsuda ◽

Y Takeuchi ◽

S Habu ◽

H Yagita ◽

...

Keyword(s):

Monoclonal Antibody ◽

Fetal Liver ◽

Critical Role ◽

In Utero ◽

In Vitro Studies ◽

Specific Interactions ◽

Erythroid Progenitors

Hematopoiesis requires specific interactions with the microenvironments, and VLA-4 has been implicated in these interactions based on in vitro studies. To study the role of VLA-4 in hematopoiesis in vivo, we performed in utero treatment of mice with an anti-VLA-4 monoclonal antibody. Although all hematopoietic cells in fetal liver expressed VLA-4, the treatment specifically induced anemia. It had no effect on the development of nonerythroid lineage cells, including lymphoids and myeloids. In the treated liver almost no erythroblast was detected, whereas the erythroid progenitors, which give rise to erythroid colonies in vitro, were present. These results indicate that VLA-4 plays a critical role in erythropoiesis, while it is not critical in lymphopoiesis in vivo.

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The Role of K-ras Signaling in Erythropoiesis In Vivo.

Blood ◽

10.1182/blood.v106.11.3136.3136 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3136-3136

Author(s):

Jing Zhang ◽

Yangang Liu ◽

Caroline Beard ◽

Rudolf Jaenisch ◽

Tyler Jacks ◽

...

Keyword(s):

Fetal Liver ◽

Erythroid Differentiation ◽

Liver Cells ◽

Ras Signaling ◽

Erythroid Progenitors ◽

Akt Activation ◽

Fetal Liver Cells ◽

Terminal Erythroid Differentiation

Abstract K-ras plays an important role in hematopoiesis. K-ras-deficient mouse embryos die around E12-E13 with severe anemia. In humans, oncogenic mutations in K-ras gene are identified in ~30% of patients with acute myeloid leukemia. We used mouse primary erythroid progenitors as a model system to study the role of K-ras signaling in vivo. Both Epo- and stem cell factor (SCF) - dependent Akt activation are greatly reduced in K-ras-/- fetal liver cells, whereas other cytokine- induced pathways, including Stat5 and p44/p42 MAP kinase, are activated normally. The reduced Akt activation in erythroid progenitors per se leads to delayed erythroid differentiation. Our data identify K-ras as the major regulator for cytokine-dependent Akt activation, which is important for erythroid differentiation in vivo. Overexpression of oncogenic Ras in primary fetal erythroid progenitors led to their continual proliferation and a block in terminal erythroid differentiation. Similarly, we found that primary fetal liver cells expressing oncogenic K-ras from its endogenous locus undergo abnormal proliferation and terminal erythroid differentiation is partially blocked. We are currently investigating the signal transduction pathways activated by this oncogenic K-ras that underlies these cellular phenotypes.

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Abnormal Distribution of Erythroid Progenitors Populations in Mice Lacking p45 NF-E2.

Blood ◽

10.1182/blood.v114.22.1988.1988 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1988-1988

Author(s):

Jadwiga Gasiorek ◽

Gregory Chevillard ◽

Zaynab Nouhi ◽

Volker Blank

Keyword(s):

Bone Marrow ◽

Red Blood Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Globin Genes ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Adult Mice ◽

Deficient Mice

Abstract Abstract 1988 Poster Board I-1010 The NF-E2 transcription factor is a heterodimer composed of a large hematopoietic-specific subunit called p45 and widely expressed 18 to 20-kDa small Maf subunits. In MEL (mouse erythroleukemia) cells, a model of erythroid differentiatin, the absence of p45 is inhibiting chemically induced differentiation, including induction of globin genes. In vivo, p45 knockout mice were reported to show splenomegaly, severe thrompocytopenia and mild erythroid abnormalities. Most of the mice die shortly after birth due to haemorrhages. The animals that survive display increased bone, especially in bony sites of hematopoiesis. We confirmed that femurs of p45 deficient mice are filled with bone, thus limiting the space for cells. Hence, we observed a decrease in the number of hematopoietic cells in the bone marrow of 3 months old mice. In order to analyze erythroid progenitor populations we performed flow cytometry using the markers Ter119 and CD71. We found that p45 deficient mice have an increased proportion of early erythroid progenitors (proerythroblasts) and a decreased proportion of late stage differentiated red blood cells (orthochromatic erythroblasts and reticulocytes) in the spleen, when compared to wild-type mice. We showed that the liver of p45 knockout adult mice is also becoming a site of red blood cell production. The use of secondary sites, such as the spleen and liver, suggests stress erythropoiesis, likely compensating for the decreased production of red blood cells in bone marrow. In accordance with those observations, we observed about 2 fold increased levels of erythropoietin in the serum of p45 knockout mice.Overall, our data suggest that p45 NF-E2 is required for proper functioning of the erythroid compartment in vivo. Disclosures: No relevant conflicts of interest to declare.

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Control of Hemoglobin Switching by BCL11A.

Blood ◽

10.1182/blood.v114.22.5.5 ◽

2009 ◽

Vol 114 (22) ◽

pp. 5-5

Author(s):

Jian Xu ◽

Vijay G. Sankaran ◽

Yuko Fujiwara ◽

Stuart H. Orkin

Keyword(s):

Gene Silencing ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Association Studies ◽

Globin Gene ◽

Clinical Severity ◽

Genome Wide Association Studies ◽

Erythroid Cells ◽

B Locus

Abstract Abstract 5 All vertebrates switch expression of globin chains during development. In humans b-like globins switch from embryonic to fetal to adult, whereas in the mouse a single switch from embryonic to adult occurs. The switch from human fetal (g) to adult (b) expression is especially critical in the b-hemoglobin disorders, such as sickle cell anemia and the b-thalassemias. Delay of the switch or reactivation of the fetal gene in the adult stage greatly ameliorates clinical severity. Despite intensive molecular studies of the human b-globin cluster over more than two decades, the proteins regulating the switch, and the mechanisms controlling the process, have been largely elusive. Recently, genome-wide association studies identified genetic variation at a chromosome 2 locus that correlates with the level of HbF in different populations. The most highly associated single nucleotide polymorphisms (SNPs) reside in an intron of the BCL11A gene, which encodes a zinc-finger repressor protein. Previously we showed that shRNA-mediated ex vivo knockdown of BCL11A in cultured human CD34-derived erythroid precursors leads to robust HbF expression, consistent with a role for BCL11A in maintaining g-genes in a silenced state in adult cells. To address in vivo roles of BCL11A either in development or in globin gene silencing in an intact individual, we have employed stringent genetic tests of function in mice that carry a complete human b-globin gene cluster as a yeast artificial chromosome transgene (b-locus mice). Knockout of BCL11A in mice leads to failure to silence the endogenous b-like embryonic genes in adult erythroid cells of the fetal liver (>2500-fold derepression). The ratio of human g to b globin RNA in the fetal liver of BCL11A knockout mice is inverted compared to controls, such that g constitutes >90% of the b-like human expression at embryonic day (E)14.5 and >75% at E18.5. These quantitatively striking findings indicate that BCL11A controls developmental silencing of g-globin gene expression. To address by formal genetics the contribution of BCL11A to g silencing in adult animals we have employed conditional inactivation of BCL11A through hematopoietic- and erythroid-specific Cre-alleles. These experiments reveal that BCL11A is also required in vivo for g-gene silencing in adults. We observed that human g-globin expression is persistently derepressed >2000-fold (as compared to littermate controls) in bone marrow erythroblasts of 15-20 week old b-locus mice upon erythroid-specific deletion of BCL11A. Taken together, these findings establish BCL11A as the first genetically validated transcriptional regulator of both developmental control of globin switching and silencing of g-globin expression in adults. The recognition of these roles for BCL11A now permits focused mechanistic studies of the switch. In human erythroid cells, BCL11A physically interacts with at least two corepressor complexes, Mi-2/NuRD and LSD1/CoREST, as well as the erythroid transcription factor GATA-1 and the HMG-box protein SOX6. Rather than binding to the promoters of the g- or b-globin genes as do these latter factors, BCL11A protein occupies the upstream locus control and g-d-intergenic regions of the b-globin cluster (as determined by high resolution ChIP-Chip analysis), suggesting that BCL11A mediates long-range interactions and/or reconfigures the locus during different stages. An in-depth mechanistic understanding of globin switching offers the prospect for design of target-based activation of HbF in adult erythroid cells of patients with hemoglobin disorders. Disclosures: No relevant conflicts of interest to declare.

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Polymeric Immunoglobulin A1 Controls Erythroblast Proliferation and Accelerates Erythropoiesis Recovery in Anemia

Blood ◽

10.1182/blood.v118.21.679.679 ◽

2011 ◽

Vol 118 (21) ◽

pp. 679-679

Author(s):

Séverine Coulon ◽

Michael Dussiot ◽

Damien Grapton ◽

Thiago Maciel ◽

Pamella Huey Mei Wang ◽

...

Keyword(s):

Cell Activation ◽

Conflicts Of Interest ◽

Iga Deficiency ◽

Mitogen Activated Protein Kinase ◽

Erythroid Progenitors ◽

Humanized Mouse Model ◽

Stress Erythropoiesis ◽

J Chain ◽

Mitogen Activated Protein ◽

Clonogenic Potential

Abstract Abstract 679 Erythropoietin (Epo) is the main cytokine controlling terminal erythropoiesis. Twenty years ago a model of erythropoiesis regulation has been described suggesting that the circulating Epo level is suboptimal and only a fraction of erythroid progenitors survives to produce erythrocytes (Koury and Bondurant; Science, 1990). Therefore differential Epo sensitivity would control erythroblasts survival. However, cell autonomous and/or environmental factors controlling this process remain elusive. Erythroblasts highly express the transferrin receptor 1 (TfR1), which has been shown to be essential for iron uptake allowing hemoglobin synthesis. We have previously identified TfR1 as an unsuspected polymeric (p)IgA1 receptor. Here we show that TfR1-bound pIgA1 (but not monomeric (m)IgA1) rescued the growth and clonogenic potential of human erythroblasts under suboptimal Epo concentrations. In a humanized mouse model (α1KI mice) IgA1 polymers increased spleen homeostatic erythropoietic activity and accelerated stress erythropoiesis in several models of anemia (phenylhydrazine and autoimmune hemolytic anemia; 5-fluorouracyl-induced central anemia). By contrast, mice lacking the immunoglobulin joining-(J) chain (α1KI/J-chain−/−), which are devoid of pIgA1 demonstrated a delayed recovery from anemia. Moreover, IgA deficiency patients presented increased Epo levels. Upon hypoxia, pIgA1 levels increased in both humans and mice. Likewise, α1KI mice submitted to chronic intermittent hypoxia presented an enhanced erythroblasts expansion relative to their LT controls. Therefore, hypoxia regulates pIgA1 production and, in turn, pIgA1/TfR1 interaction modulates erythroblast sensitivity to growth factors by decreasing cell activation threshold. We also showed that the natural TfR1 ligand, iron-loaded transferrin (Fe-Tf), also accelerated recovery from acute anemia. At the molecular level, pIgA1 and Fe-Tf converged for Epo-dependent erythroblasts proliferation. TfR1 engagement increased cell sensitivity to Epo by inducing activation of mitogen-activated protein kinase and phosphatidylinositol-3-kinase signaling pathways. These cellular responses were mediated by the TfR1-internalization motif, YXXΦ. Therefore, pIgA1 and TfR1 are positive regulators of erythropoiesis in both physiological and pathological situations. This study shed light on a new role of TfR1 as a signaling competent receptor. Targeting this pathway could provide alternative approaches to treat anemia, in particular in Epo hypo-responsive patients and dyserythropoiesis. Disclosures: No relevant conflicts of interest to declare.

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Lncrna Hematlas Defines Blood Lineage-Specific RNA Expression Signatures and Novel Lincrna Biomarkers

Blood ◽

10.1182/blood.v122.21.3669.3669 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3669-3669

Author(s):

Stephan Emmrich ◽

Franziska Schmidt ◽

Ramesh Chandra Pandey ◽

Aliaksandra Maroz ◽

Dirk Reinhardt ◽

...

Keyword(s):

Stem Cells ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Gene Set Enrichment Analysis ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Lncrna Expression ◽

Fold Reduction ◽

Non Coding Rnas

Abstract Long non-coding RNAs (lncRNAs) recently emerged as central regulators of chromatin and gene expression. We created a comprehensive lncRNA HemAtlas in human and murine blood cells. We sampled RNA from differentiated granulocytes, monocytes, erythroid precursors, in vitro maturated megakaryocytes, CD4-T and CD8-T cells, NK cells, B cells and stem cells (human CD34+ cord blood hematopoietic stem and progenitor cells [CB-HSPCs]) and subjected them to microarray analysis of mRNA and lncRNA expression. Moreover, the human LncRNA HemAtlas was complemented with human hematopoietic stem cells (HSCs; CD34+/CD38-), megakaryocytic/erythroid progenitors (MEPs; CD34+/CD38+/CD45RA-/CD123-), common myeloid progenitors (CMPs; CD34+/CD38+/CD45RA-/CD123+) and granulocytic/monocytic progenitors (GMPs; CD34+/CD38+/CD45RA+/CD123+) from fetal liver (FL), CB and peripheral blood (PB) HSPCs. The complete microarray profiling of the differentiated cells yielded a total of 1588 (on Arraystar® platform) and 1439 lncRNAs (on NCode® platform), which were more than 20-fold differentially expressed between the blood lineages. Thus, a core fraction of lncRNAs is modulated during differentiation. LncRNA subtype comparison for each lineage, schematics of mRNA:lncRNA lineage coexpression and genomic loci correlation revealed a complex genetic interplay regulating hematopoiesis. Integrated bioinformatic analyses determined the top 50 lineage-specific lncRNAs for each blood cell lineage in both species, while gene set enrichment analysis (GSEA) confirmed lineage identity. The megakaryocytic/erythroid expression program was already evident in MEPs, while monocytoc/granulocytic signatures were found in GMPs. Amongst all significantly associated genes, 46% were lncRNAs, while 5% belonged to the subgroup of long intervening non-coding RNAs (lincRNA). For human megakaryocytes, erythroid cells, monocytes, granulocytes and HSPCs we validated four lincRNA candidates, respectively, to be specifically expressed by qRT-PCR. RNAi knock-down studies using two shRNA constructs per candidate demonstrated an impact on proliferation, survival or lineage specification for at least one specific lincRNA per lineage. We detected a 3 to 4.5-fold increased colony-forming capacity upon knockdown of the HSPC-specific PTMAP6 lincRNA in methylcellulose colony-forming unit (CFU) assays. Inversely, knockdown of monocyte-specific DB519945 resulted in 3.5 to 5.5-fold reduction of the total number of CFUs. Likewise, the total CFU counts was 4.3-fold reduced upon knockdown of megakaryocyte-specific AK093872. Kockdown of the granulocyte-specific LINC00173 perturbed granulocytic in vitro differentiation as assessed by the percentage of CD66b+/CD13+ granulocytes (2-fold reduction) and nuclear lobulation (MGG-stained cytospins). The erythroid-specific transcript AY034471 showed 25 to 50% reduction in burst-forming units in collagen-based assays. Thus, our study provides a global human hematopoietic lncRNA expression resource and defines blood-lineage specific lncRNA marker and regulator genes. Disclosures: No relevant conflicts of interest to declare.

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